A nucleic acid-based strategy for highly specific discrimination between mutant and wild-type sequences

Abstract

This study presents a novel Terminal Self-Competitive Nucleic Acid Probe (TSCP) for rapid, precise detection of mutant and wild-type gene sequences. The TSCP probe employs overlapping binding domains and inhibition strands, enabling highly specific competitive hybridization. Combined with hybridization chain reaction (HCR) for signal amplification, the method achieves nanomolar sensitivity without requiring proteases, temperature control, or complex instrumentation. Experimental validation in blood samples demonstrated distinct fluorescent signals for mutant and wild-type sequences, allowing accurate target differentiation. The probe is cost-effective and scalable, as it only requires nucleic acid synthesis for new mutations, ensuring rapid adaptation to emerging variants. This straightforward, versatile approach facilitates simultaneous detection of genetic variations, making it highly suitable for molecular diagnostics and genetic research. Its high specificity, rapid response, and low-cost production underscore its potential as a practical tool for advancing mutation detection in clinical and research settings. By eliminating the need for specialized equipment and enabling quick deployment, the TSCP-based method offers a widely accessible solution for identifying genetic mutations, enhancing both diagnostic accuracy and research efficiency.